Summary: Anthrax is a severe disease caused by the gram-positive bacterium Bacillus anthracis. The organism infects humans and many other animals and the only identified virulence factors are anthrax toxin and a glutamic acid capsule, both of which are encoded on large plasmids. Inhalation anthrax, the most severe form of the disease, is a systemic infection in which the organism spreads to the lymph nodes and then into the blood where it is able to replicate to levels as high as 100 million bacteria / ml. Factors encoded on the chromosome have also been implicated in virulence, since certain strains lacking the virulence plasmids are still capable of causing disease in immunized animals. The future development of these "vaccine resistant" strains as bioterrorism weapons is of major concern, and stresses the importance of establishing a fundamental understanding of the molecular mechanisms involved in the pathogenesis of this bacterium. For many bacterial pathogens, the ability to survive in the human host requires the acquisition of the essential element iron. Mechanisms involved in the acquisition of iron have been shown to be important for the virulence of numerous bacterial pathogens, including organisms that replicate in the blood, where much of the available iron is sequestered by host iron compounds such as transferrin and hemoglobin (in erythrocytes). No investigations have examined the mechanism by which B. anthracis acquires iron during growth in vivo or in vitro. This is an important area of study that has not been explored, and in this proposed research, I intend to test the hypothesis that virulence factors involved in the transport and utilization of host iron compounds are present in B. anthracis. Experimental Design We will first determine if B. anthracis can utilize host compounds, such as heme, hemoglobin, transferrin or lactoferrin as iron sources. If B. anthracis is able to use some of these compounds, we will search the partially completed genomic sequence of B. anthracis to identify genes that may be involved in the acquisition of iron from host compounds. Systems that will be searched for include high-affinity siderophore (or inorganic iron) transport systems, transferrin or lactoferrin binding proteins and heme or hemoglobin utilization systems. Since genetic tools are available in B. anthracis, mutations in the genes encoding these transport systems will be constructed. These mutant strains of B. anthracis will then be tested to confirm that these genes are important in vitro for iron acquisition and then examined in relevant cell lines and/or animal models to determine if these genes affect virulence.